Signal amplification in plasmonic specific-binding partner assays

ABSTRACT

The present invention relates to analyte detection devices and methods of using such devices to detect minute quantities of a target analyte in a sample. In particular, the invention provides an analyte detection device comprising a plurality of composite metallic nanostructures conjugated to analyte binding partners and a surface containing a metallic nanolayer on which a plurality of capture molecules is immobilized. Methods of preparing composite nanostructures are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/037,071, filed Aug. 13, 2014 and U.S. Provisional Application No.62/082,468, filed Nov. 20, 2014, each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to systems and methods for detectingtarget analytes in a sample. In particular, the present inventionprovides a local plasmon resonance-based analyte detection systemcapable of detecting a minute quantity of a target analyte in a sample.

BACKGROUND OF THE INVENTION

Current immunoassays and biomolecule binding assays typically requiremultiple steps and sophisticated equipment to perform the assays. Thelack of sensitivity and the complexity involved in performing suchheterogeneous assays arises from the specific need to separate labeledfrom unlabeled specific binding partners.

Attempts to develop assays based on the local surface plasmon resonance(LSPR) properties of noble metal nanoparticles have been made (Tokel etal., Chem Rev., Vol. 114: 5728-5752, 2014). LSPR is the collectiveoscillation of electrons in nanometer-sized structures induced byincident light. Metallic nanoparticles have a strong electromagneticresponse to refractive index changes in their immediate vicinity andthus shifts in the resonance frequency of the nanoparticles can bemeasured as an indicator of molecules binding to the nanoparticlesurface. Although metallic nanoparticles, particularly goldnanoparticles, have been employed in diagnostic assays to detect bindingevents, such assays generally suffer from low sensitivity and cannot beused to quantitatively monitor the kinetics of sequential bindingevents.

Thus, improved assay methods employing a homogenous format whileproviding increased sensitivity are needed. Assays utilizing standardlaboratory techniques, such as spectroscopy, would also be desirable.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that compositemetallic nanostructures can enhance the optical signals induced bybinding of a molecule to a metallic nanolayer surface. The amplificationobserved greatly increases the sensitivity of detecting specificbiomolecular binding events such that sub-picogram quantities of thebiomolecule can be detected. Accordingly, the present invention providesanalyte detection devices and methods of using such devices to detectminute quantities of a target analyte in a sample.

In one embodiment, the analyte detection devices comprise a plurality ofdetection conjugates, a surface containing a metallic nanolayer, and aplurality of capture molecules, wherein the capture molecules areimmobilized on the metallic nanolayer and are capable of specificallybinding to the target analyte. In embodiments in which the analytedetection devices are configured in a sandwich assay format, thedetection conjugates comprise composite metallic nanostructures coupledto binding partners that are capable of specifically binding to thetarget analyte. In embodiments in which the analyte detection devicesare configured in a direct competitive assay format, the detectionconjugates composite metallic nanostructures coupled to target analytes.

The composite metallic nanostructures in the detection conjugatesgenerally comprise at least two noble metals, transition metals, alkalimetals, lanthanides, or combinations thereof. In some embodiments, thecomposite metallic nanostructures comprise at least two metals selectedfrom gold, silver, copper, platinum, palladium, cadmium, iron, nickel,and zinc. In certain embodiments, each of the composite metallicnanostructures comprises a core of a first metal and a coating of asecond metal. In some embodiments, the core may be silver or copper witha gold coating. In other embodiments, the core of a first metal may bedissolved following coating such that a hollow structure comprised ofthe second coating metal results.

The metallic nanolayer deposited on the surface can be a metallic filmor comprised of a plurality of metallic nanostructures immobilized onthe surface. The metallic nanolayer can also be comprised of a noble ortransition metal. In some embodiments, the metallic nanolayer comprisesgold, silver, copper, platinum, palladium, cadmium, zinc or a compositethereof. In one embodiment, the metallic nanolayer comprises gold. Inanother embodiment, the metallic nanolayer comprises silver. In stillanother embodiment, the metallic nanolayer comprises a silver nanolayeroverlaid with a gold nanolayer.

The present invention also provides methods of detecting a targetanalyte in a sample using the analyte detection devices describedherein. In one embodiment, the methods comprise mixing the sample with aplurality of detection conjugates, contacting the mixture with a surfacecontaining a metallic nanolayer on which a plurality of capturemolecules are immobilized, exposing the surface to a light source at awavelength range within the ultraviolet-visible-infrared spectrum; andmeasuring an optical signal from the surface, wherein a change in theoptical signal indicates the presence of the target analyte in thesample. In certain embodiments, the methods of the present invention arecapable of detecting femtogram to nanogram quantities of a targetanalyte in sample.

The present invention includes an assay complex comprising a detectionconjugate comprising a composite metallic nanostructure coupled to abinding partner; a target analyte; and a metallic nanolayer-coated beadon which a capture molecule is immobilized, wherein the binding partnerin the detection conjugate is bound to a first epitope on the targetanalyte and the capture molecule is bound to a second epitope on thetarget analyte, thereby forming a complex comprising the detectionconjugate, target analyte, and the capture molecule. In someembodiments, the composite metallic nanostructure is a gold-coatedsilver nanostructure or a gold-coated copper nanostructure and themetallic nanolayer coating on the bead comprises gold.

In another aspect, the present invention provides a method for preparingcomposite metallic nanostructures for use in the detection devices andmethods described herein. In one embodiment, the methods comprisepreparing a first solution comprising a mixture of a polymer andchloroauric acid, preparing a second solution comprising silver orcopper nanostructures, and incubating the first solution with the secondsolution for a period of time, wherein the resulting mixture comprisesgold-coated silver nanostructures or gold-coated copper nanostructures.In certain embodiments, a reducing agent, such as ascorbic acid, isadded to the reaction mixture to increase the quantity of nanostructuresproduced. In one embodiment, the polymer in the first solution ispolyvinylpyrrolidone. In another embodiment, the polymer in the firstsolution is polyvinyl alcohol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Plot of shift in peak wavelength versus acquisition time for abovine serum albumin (BSA)-coupled gold nanolayer sensor (channel 1) andhuman IgG-coupled gold nanolayer sensors (channels 2-4). Arrows indicatethe injection sequence and concentration of unlabeled protein A, 1 mMHCl, or protein A labeled with colloidal gold (CGC).

FIG. 2. Plot of shift in peak wavelength versus acquisition time for ananti-CRP C7 antibody-coupled gold nanolayer sensor. Arrows indicateinjection sequence of 0 to 100 ng/ml concentrations of CRP in differentchannels (CRP loading), 1 μg/ml unlabeled anti-CRP C6 antibody, or 3μg/ml anti-CRP C6 antibody labeled with colloidal gold (C6-CGC). Nofurther C6-CGC binding was observed when sensor surface was occupiedwith unlabeled anti-CRP C6 antibody.

FIG. 3. Plot of shift in peak wavelength versus acquisition time for ananti-CRP C7 antibody-coupled gold nanolayer sensor. Arrows indicate theinjection sequence of 0 to 100 ng/ml concentrations of CRP in differentchannels (CRP loading), 1 μg/ml anti-CRP C6 antibody labeled withcolloidal gold (C6-CGC), 3 μg/ml C6-CGC, or 1 mM HCl (Acid).

FIG. 4, panel A. Reflectance spectra of anti-CRP C7 antibody-coupledgold nanolayer sensors loaded with 10 ng/ml CRP at the various C6-CGCconcentrations in FIG. 3.

FIG. 4, panel B. Plot of shift in peak wavelength versus acquisitiontime for an anti-CRP C7 antibody-coupled gold nanolayer sensor incubatedwith one of three concentrations of CRP following introduction of 3μg/ml anti-CRP C6 antibody labeled with colloidal gold (C6-CGC). Thetable at the right depicts peak analysis 700 seconds after introductionof C6-CGC.

FIG. 5. Plot of shift in peak wavelength versus acquisition time for ananti-CRP C7 antibody-coupled gold nanolayer sensor. Arrows indicate theinjection sequence of 0 to 100 ng/ml concentrations of CRP in differentchannels (CRP loading with incubation time minimized), 3 μg/ml anti-CRPC6 antibody labeled with colloidal gold (C6-CGC), or 1 mM HCl (Acid).

FIG. 6. Plot of shift in peak wavelength versus acquisition time fortraces in FIG. 5 following immediate introduction of 3 μg/ml anti-CRP C6antibody labeled with colloidal gold (C6-CGC). The table at the rightdepicts peak analysis 700 seconds after introduction of C6-CGC ascompared to the peak shifts obtained with CRP incubation (values shownin panel B (bottom panel) of FIG. 4).

FIG. 7. Plot of shift in peak wavelength versus acquisition time for ananti-CRP C7 antibody-coupled gold nanolayer sensor incubated with one ofthree concentrations of CRP and C6 anti-CRP antibody conjugated togold-coated silver nanostructures. The control was a gold nanolayersensor with immobilized bovine serum albumin (BSA) in place of C7antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery thatsignificant amplification in LSPR-based assays can be achieved withcomposite metallic nanostructure-labeled binding partners. Thus, thepresent invention provides analyte detection devices comprising anLSPR-surface, e.g., a surface containing a metallic nanolayer, aplurality of capture molecules immobilized to the metallic nanolayer,and a plurality of detection conjugates comprising composite metallicnanostructures coupled to biomolecules.

The analyte detection devices can be configured in a sandwich assayformat or a direct competitive assay format. For instance, in oneembodiment, an analyte detection device in a sandwich assay formatcomprises (i) a plurality of detection conjugates, wherein theconjugates comprise composite metallic nanostructures coupled to bindingpartners that are capable of specifically binding to a target analyte,(ii) a surface containing a metallic nanolayer, and (iii) a plurality ofcapture molecules, wherein the capture molecules are immobilized on themetallic nanolayer and are capable of specifically binding to the targetanalyte. In another embodiment, an analyte detection device in a directcompetitive assay format comprises (i) a plurality of detectionconjugates, wherein the conjugates comprise composite metallicnanostructures coupled to target analytes, (ii) a surface containing ametallic nanolayer, and (iii) a plurality of capture molecules, whereinthe capture molecules are immobilized on the metallic nanolayer and arecapable of specifically binding to the target analytes.

The analyte detection devices of the invention comprise a surfacecontaining a metallic nanolayer. The surface can be any suitable sizeand shape, such as a chip, a well, a cuvette, or a bead. In someembodiments, the surface is a rectangular chip. In other embodiments,the surface is a disc. In certain embodiments, the surface is thebottom, the cover, and/or interior walls of a cuvette (e.g., cylindricalor rectangular cuvette). In still other embodiments, the surface is anarray of non-metallic particles. The surface can be manufactured fromvarious materials including, but not limited to, glass, quartz, silicon,silica, polystyrene, graphite, fabric (e.g. polyethylene fabrics), mesh,or a membrane (e.g. latex, polyvinyl, nylon, or polyester membranes).

A metallic nanolayer is preferably deposited on the surface. Themetallic nanolayer may, in some embodiments, cover the entire surfacearea of the particular surface. In other embodiments, the metallicnanolayer may be deposited only on a portion of the surface. Forexample, the surface may contain a plurality of depressions or wells andthe metallic nanolayer is deposited within the depressions or wells. Inother embodiments, the metallic nanolayer may be applied to the surfaceas a plurality of spaced deposits across the surface. The opticalproperties of the metallic nanolayer can be adjusted by varying thethickness of the nanolayer and/or the nature of nanostructures. In oneembodiment, the nanolayer is comprised of metallic nanoislands. Inanother embodiment, the nanolayer is comprised of nanorods. Suitablethicknesses of the metallic nanolayer for use in the devices and methodsof the invention include from about 0.5 nm to about 100 nm, about 5 nmto about 30 nm, or about 3 nm to about 10 nm. Exemplary surfaces with ametallic nanolayer coating that can be used in the devices and methodsof the invention include the surfaces described in U.S. PatentPublication No. 2006/0240573, which is hereby incorporated by referencein its entirety.

In certain embodiments, the metallic nanolayer is a metallic film.Methods of depositing metallic films on a substrate surface are known tothose of skill in the art and include, but are not limited to, atomiclayer deposition, pulsed laser deposition, drop casting, vapordeposition, and adsorption. See, e.g., Atanasov et al., Journal ofPhysics: Conference Series 514 (2014); Walters and Parkin, Journal ofMaterials Chemistry, 19: 574-590, 2009; and Gupta et al., J. Appl. Phys.92, 5264-5271, 2002, each of which is herein incorporated by referencein its entirety. The metallic film may comprise other components, e.g.the metallic film may be a polymer film, a Langmuir-Blodgett film or anoxide film. In some embodiments, the metallic film comprises two layers,wherein each layer comprises a different metal. By way of example, themetallic film may comprise a silver layer overlaid with a gold layer.

In other embodiments, the metallic nanolayer comprises a plurality ofmetallic nanostructures immobilized to the surface. Metallicnanostructures can be immobilized to the surface by treating the surfacematerial with a reagent to add functional chemical groups, such ascyanide, amine, thiols, carboxyl, aldehyde or maleimide, and reactingthe metallic nanostructures with the treated surface. Metallicnanostructures are known to bind to such functional chemical groups withhigh affinity. In some embodiments, the metallic nanostructurescomprising the metallic nanolayer are spherical nanoparticles. Suchnanoparticles have diameters that are less than about 300 nm, less thanabout 200 nm, or less than about 150 nm. In some embodiments, thespherical nanoparticles have a diameter from about 5 nm to about 200 nm,from about 10 nm to about 100 nm, or from about 20 nm to about 60 nm. Incertain embodiments, the size of the metallic nanostructures used tocreate the metallic nanolayer are similar to the size of the compositenanostructures used in the detection conjugates. In such embodiments,matching the size of the two sets of nanostructures can provide anoptimal wavelength shift in a reflectance, emission or scatteringspectrum.

The metallic nanolayer (metallic film or plurality of metallicnanostructures) may be composed of a noble metal or composite thereof.In other embodiments, the metallic nanolayer (metallic film or pluralityof metallic nanostructures) may be composed of a transition metal orcomposite thereof. In certain embodiments, the metallic nanolayercomprises a metal selected from gold, silver, copper, platinum,palladium, ruthenium, rhodium, osmium, iridium, titanium, chromium,cadmium, zinc, iron, cobalt, nickel, and composites thereof. In oneparticular embodiment, the metallic nanolayer (e.g. metallic film orplurality of metallic nanostructures) comprises gold. In anotherparticular embodiment, the metallic nanolayer (e.g. metallic film orplurality of metallic nanostructures) comprises silver. In certainembodiments, the metallic nanolayer (e.g. metallic film or plurality ofmetallic nanostructures) comprises a composite of gold and silver orgold and copper. Use of alkali metals (e.g., lithium, sodium, potassium,rubidium, cesium, and francium) or lanthanides (e.g., lanthanum, cerium,praseodymium, neodymium, promethium, samarium, europium, gadolinium,terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium)may improve intensity of the LSPR peaks. Accordingly, in someembodiments, the metallic nanolayer (metallic film or plurality ofmetallic nanostructures) may be composed of one or more alkali metals orlanthanides. In other embodiments, the metallic nanolayer (metallic filmor plurality of metallic nanostructures) may be composed of acombination of a noble metal and an alkali metal or lanthanide.

The analyte detection devices of the invention further comprise aplurality of capture molecules immobilized to the metallic nanolayerdeposited on a surface. The capture molecules are capable ofspecifically binding to a target analyte. As used herein, “specificbinding” refers to binding to a target molecule with high affinity,e.g., an affinity of at least 10⁻⁶M. In some embodiments, the capturemolecules are haptens and other small molecules, drugs, hormones,biological macromolecules including, but not limited to, antibodies orfragments thereof (e.g., Fv, Fab, (Fab)₂, single chain, CDR etc.),antigens, receptors, ligands, polynucleotides, aptamers, polypeptides,polysaccharides, lipopolysaccharides, glycopeptides, lipoproteins, ornucleoproteins. In certain embodiments, the plurality of capturemolecules are antibodies. In other embodiments, the plurality of capturemolecules are antigens.

Methods of immobilizing molecules to metallic nanolayers ornanostructures are known to those of skill in the art. Such methodsinclude conjugation chemistries, such as those involving1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC),sulfo-NHS coupling, hydrophobic binding or thioether chemistry. In someembodiments, the molecule can be coupled to the metallic nanolayer ornanostructure indirectly through a larger carrier molecule or protein.Such indirect coupling is particularly useful when the molecule issmall, such as a hormone, a drug, and other small molecules less than 10kD. Preferably, the carrier protein is not capable of specificinteraction with the target analyte.

The analyte detection devices of the invention may also comprise aplurality of detection conjugates. Detection conjugates comprisemetallic nanostructures coupled to binding partners capable ofspecifically binding to a target analyte or the capture moleculesdepending on the assay configuration. For example, in embodiments inwhich the device is configured in a sandwich assay format, the detectionconjugates comprise metallic nanostructures coupled or conjugated tobinding partners that are capable of specifically binding a targetanalyte. In other embodiments in which the device is configured in adirect competitive assay format, the detection conjugates comprisemetallic nanostructures coupled or conjugated to target analytes.

The binding partners can be the same types of molecules as the capturemolecules, including, but not limited to haptens and other smallmolecules, drugs, hormones, biological macromolecules such as antibodiesor fragments thereof (e.g., Fv, Fab, (Fab)₂, single chain, CDR etc.),antigens, receptors, ligands, polynucleotides, aptamers, polypeptides,polysaccharides, lipopolysaccharides, glycopeptides, lipoproteins, ornucleoproteins. In some embodiments, the binding partners are the sametype of molecule as the capture molecules, but preferably bind to thetarget analyte at a location distinct from the binding site of thecapture molecules. By way of example, the binding partners and thecapture molecules can both be antibodies that recognize a targetanalyte, but the epitope to which the binding partners bind the targetanalyte is separate from and ideally non-overlapping with the epitope towhich the capture molecules bind the target analyte. Thus, in certainembodiments, the binding partners are antibodies that recognize a firstepitope of a target analyte and the capture molecules are differentantibodies that recognize a second epitope of a target analyte.

The metallic nanostructures in the detection conjugates can be composedof a noble metal or composite thereof. In some embodiments, the metallicnanostructures in the detection conjugates may be composed of atransition metal or composite thereof. In some embodiments, the metallicnanostructures in the detection conjugates may comprise an alkali metalor lanthanide in combination with a noble or transition metal. Incertain embodiments, metallic nanostructures in the detection conjugatescomprise a metal selected from gold, silver, copper, platinum,palladium, ruthenium, rhodium, osmium, iridium, titanium, chromium,cadmium, zinc, iron, cobalt, nickel, and composites thereof. In oneembodiment, the metallic nanostructures are gold nanostructures. Inanother embodiment, the metallic nanostructures are silvernanostructures.

In preferred embodiments, the metallic nanostructures in the detectionconjugates are composite metallic nanostructures. “Composite metallicnanostructures” refers to nanostructures that comprise at least twonoble metals, transition metals, alkali metals, or lanthanides. The twoor more metals may be mixed together, as in an alloy, or the two or moremetals may be present in separate portions of the nanostructure. Forexample, one metal may form the core of the nanostructure, whereas thesecond metal forms an outer shell or coating of the nanostructure. Insome embodiments, the composite metallic nanostructures comprise atleast two metals selected from gold, silver, copper, platinum,palladium, ruthenium, rhodium, osmium, iridium, titanium, chromium,cadmium, zinc, iron, cobalt, and nickel. In other embodiments, thecomposite metallic nanostructures comprise at least two metals selectedfrom gold, silver, copper, platinum, palladium, cadmium, iron, nickel,and zinc. In one particular embodiment, the composite metallicnanostructures comprise gold and silver. In another embodiment, thecomposite metallic nanostructures comprise gold and copper. In yetanother embodiment, the composite metallic nanostructures comprisesilver and copper.

In some embodiments, each of the composite metallic nanostructures is analloy of a first metal and a second metal. In certain embodiments, eachof the composite metallic nanostructures comprises a core of a firstmetal and a coating of a second metal. In one embodiment, the core issilver and the coating is gold. In another embodiment, the core iscopper and the coating is gold. In another embodiment, the core issilver and the coating is copper. In some embodiments, each of thecomposite metallic nanostructures comprises a dielectric core (e.g.silicon dioxide, gold sulfide, titanium dioxide, silica, andpolystyrene), a first coating of a first metal, and a second coating ofa second metal. In one particular embodiment, the core is silica, thefirst coating (i.e. inner coating) is a silver coating, and the secondcoating is a gold coating (i.e. outer coating). In another embodiment,the core is silica, the first coating (i.e. inner coating) is a coppercoating, and the second coating is a gold coating (i.e. outer coating).

In some embodiments, the core comprising a first metal is dissolvedfollowing the coating process with a second metal to create a hollowstructure comprised of the second metal. For instance, coating of asilver core with gold nanoparticles generates a gold shell around thesilver core and the silver core is subsequently dissolved or degradedresulting in the formation of a hollow nanogold shell structure.

The metallic nanostructures include spherical nanoparticles as wellnanoplates and nanoshells. Nanoplates have lateral dimensions (e.g. edgelengths) that are greater than their thickness. Nanoplates includenanodisks, nanopolygons, nanohexagons, nanocubes, nanorings, nanostars,and nanoprisms. In some embodiments, the metallic nanostructures,including the composite nanostructures, have a geometry selected fromspherical nanoparticles, pyramidal nanoparticles, hexagonalnanoparticles, nanotubes, nanoshells, nanorods, nanodots, nanoislands,nanowires, nanodisks, nanocubes, or combinations thereof. Other shapesare also possible, including irregular shapes. In certain embodiments,the size and shape of the metallic nanostructures are not uniform—i.e.the metallic nanostructures are a heterogeneous mixture of differentshapes and sizes of nanostructures.

For spherical nanoparticles, suitable diameter ranges include from about5 nm to about 200 nm, from about 10 nm to about 100 nm, and from about20 nm to about 60 nm. For nanoplates, edge lengths may be from about 10nm to about 800 nm, from about 20 nm to about 500 nm, from about to 50nm to about 200 nm, from about 30 nm to about 100 nm, or from about 10nm to about 300 nm. The thickness of the nanoplates can range from about1 to about 100 nm, from about 5 nm to about 80 nm, from about 10 nm toabout 50 nm, or from about 5 nm to about 20 nm.

In some embodiments, the nanoplates have an aspect ratio greater than 2.The aspect ratio is the ratio of the edge length to the thickness.Preferably, the nanoplates have an aspect ratio from about 2 to about25, from about 3 to about 20, from about 5 to about 10, from about 2 toabout 15, or from about 10 to about 30.

The binding partners or target analytes can be coupled or conjugated tothe metallic nanostructures (e.g. composite nanostructures) usingsimilar methods as described above for the immobilization of the capturemolecules to the metallic nanolayer. Such methods include, but are notlimited to, EDC conjugation chemistry, sulfo-NHS coupling, hydrophobicbinding or thioether chemistry. The binding partners or target analytescan be coupled to the metallic nanostructures through various chemicalfunctionalities including thiol, amine, dithiol, acrylicphosphoramidite, azide, or alkynes.

In some embodiments, the metal or metals employed in the metallicnanolayer deposited on the surface can be the same as the metal ormetals from which the metallic nanostructures in the detectionconjugates are fabricated. For example, in one embodiment, the metallicnanolayer deposited on the surface comprises a gold film or a pluralityof gold nanostructures and the detection conjugates comprise goldnanostructures. In other embodiments, the metal employed in the metallicnanolayer deposited on the surface is different from the metal or metalsused to create the metallic nanostructures in the detection conjugates.For instance, in some embodiments, the metallic nanolayer deposited onthe surface comprises a silver film or a plurality of silvernanostructures and the detection conjugates comprise goldnanostructures. In other embodiments, the metallic nanolayer depositedon the surface comprises a gold film or a plurality of goldnanostructures and the detection conjugates comprise silvernanostructures. In certain embodiments, the metallic nanolayer depositedon the surface comprises a gold film or a plurality of goldnanostructures and the detection conjugates comprise compositenanostructures. In related embodiments, the composite nanostructurescomprise gold-coated silver nanostructures. In other particularembodiments, the metallic nanolayer deposited on the surface comprises agold film or a plurality of gold nanostructures and the detectionconjugates comprise composite nanostructures comprising gold-coatedcopper nanostructures. In yet other embodiments, the metallic nanolayerdeposited on the surface comprises a gold film or a plurality of goldnanostructures and the detection conjugates comprise compositenanostructures comprising gold-coated magnetite nanostructures. In stillother embodiments, the metallic nanolayer deposited on the surfacecomprises a gold film or a plurality of gold nanostructures and thedetection conjugates comprise composite nanostructures comprising goldand an alkali metal or lanthanide.

The present invention also includes kits comprising the analytedetection devices of the invention as disclosed herein. In oneembodiment, the kit comprises (i) a surface containing a metallicnanolayer on which a plurality of capture molecules are immobilized and(ii) a composition comprising a plurality of detection conjugates asdescribed herein. In certain embodiments, the composition is packagedseparately from the surface such that it can be brought into subsequentcontact with the surface during performance of the detection methods. Insome embodiments, the composition comprising the plurality of detectionconjugates is lyophilized, for example, in the form of a pellet or bead.In related embodiments, the surface containing the metallic nanolayercan be a chip, disc, or a cuvette. In one particular embodiment, thesurface containing the metallic nanolayer is a cuvette adapted for usewith a centrifugal rotor. In such embodiments, the metallic nanolayermay be deposited on the cover, bottom and/or walls of the cuvette.

In certain embodiments, all components of the analyte detection systemsdescribed herein are contained within a centrifugal rotor or disc. Forinstance, a rotor or disc may contain one or more reaction chambers inwhich the metallic nanolayer surface containing immobilized capturemolecules and the plurality of detection conjugates are positioned. Inone embodiment, the metallic nanolayer surface is a chip located at thebottom of the reaction chamber. In another embodiment, the metallicnanolayer is deposited directly on the floor of the reaction chamber. Instill another embodiment, the metallic nanolayer surface is a bead (e.g.plastic bead) coated with the metallic nanolayer. In all suchembodiments, the capture molecules are immobilized to the metallicnanolayer surfaces. In related embodiments, the plurality of detectionconjugates is present in the form of a lyophilized composition, such asa lyophilized bead or pellet.

In alternative embodiments, capture molecules are conjugated to metallicnanostructures, which are in colloidal suspension. The plurality ofdetection conjugates is added to the suspension in the presence of atest sample. If the target analyte is present in the sample, complexformation will occur between the detection conjugates and the suspendednanostructures containing the capture molecules resulting in a change inoptical signal (e.g., shift in peak absorbance wavelength of thesuspended nanostructures).

Accordingly, in some embodiments, the kits comprise a rotor or dischaving one or more reaction chambers, wherein each reaction chambercomprises (i) a lyophilized composition comprising a plurality ofdetection conjugates as described herein and (ii) a bead coated with ametallic nanolayer, wherein a plurality of capture molecules areimmobilized to the metallic nanolayer. Such kits provide a one-stepanalyte detection assay whereby a test sample is contacted with therotor or disc, and application of a centrifugal force to the rotor ordisc delivers the test sample to the reaction chambers where the samplemixes with the plurality of detection conjugates and the metallicnanolayer-coated bead containing immobilized capture molecules. Inembodiments in which the rotor or disc contains more than one reactionchamber, the detection conjugates and capture molecules can be selectedsuch that a different analyte can be detected in each reaction chamber.These rotor-format detection devices can be configured in the sandwichassay format, the direct competitive format, or both if the rotorscomprise multiple reaction chambers.

Any of the types of metallic nanolayers or metallic nanostructuresdiscussed herein can be used with these rotor-format detection devices.In some embodiments, the metallic nanolayer coating on the bead is agold nanolayer and the metallic nanostructures in the detectionconjugates are gold nanostructures. In other embodiments, the metallicnanolayer coating on the bead is a silver nanolayer and the metallicnanostructures in the detection conjugates are gold nanostructures. Instill other embodiments, the metallic nanolayer coating on the bead is agold nanolayer and the metallic nanostructures in the detectionconjugates are silver nanostructures. In one embodiment, the metallicnanolayer coating on the bead is a silver nanolayer overlaid with a goldnanolayer and the metallic nanostructures in the detection conjugatesare gold nanostructures. In certain embodiments, the metallic nanolayercoating on the bead is a gold nanolayer and the metallic nanostructuresin the detection conjugates are composite nanostructures. For instance,in one embodiment, the composite nanostructures are gold-coated silvernanostructures. In another embodiment, the composite nanostructures aregold-coated copper nanostructures.

The kits of the invention may also include instructions for using thedevice to detect an analyte in a test sample, devices or tools forcollecting biological samples, and/or extraction buffers for obtainingsamples from solid materials, such as soil, food, and biologicaltissues.

The present invention also provides methods of detecting a targetanalyte in a sample. In one embodiment, the methods comprise (i) mixinga test sample with a plurality of detection conjugates as describedherein; (ii) contacting the mixture with a surface containing a metallicnanolayer, wherein a plurality of capture molecules as described hereinare immobilized to the metallic nanolayer; (iii) exposing the surface toa light source at a wavelength range within theultraviolet-visible-infrared spectrum; and (iv) measuring an opticalsignal from the surface, wherein a change in the optical signalindicates the presence of the target analyte in the sample.

In some embodiments, the detection methods are sandwich assays. In suchembodiments, the detection conjugates comprise metallic nanostructurescoupled to binding partners that are capable of specifically binding tothe target analyte if present in the sample to form analyte-detectionconjugate complexes. The plurality of capture molecules, which areimmobilized to the metallic nanolayer surface, are also capable ofspecifically binding to the target analyte if present in the sample. Themetallic nanolayer is exposed to a light source and an optical signal ismeasured, wherein a change in the optical signal indicates the presenceof analyte in the sample. By way of illustration, when a samplecontaining the target analyte is mixed with the plurality of detectionconjugates, the target analyte binds to the binding partners in thedetection conjugates to form analyte-detection conjugate complexes.These complexes in turn bind to the plurality of capture moleculesimmobilized to the metallic nanolayer surface through the analytethereby bringing the metallic nanostructures in the detection conjugatesin close proximity to the metallic nanolayer surface. The amount oflight that is absorbed or scattered by the metallic nanolayer surface isaffected by the proximity of the metallic nanostructures in the complexand thus produces an enhanced shift in the peak absorption wavelength,which indicates the presence of the target analyte in the sample.

In other embodiments, the detection methods are competitive assays. Insuch embodiments, the detection conjugates comprise metallicnanostructures coupled to the target analyte of interest. As in thesandwich assay method, the plurality of capture molecules, which areimmobilized to the metallic nanolayer surface, are capable ofspecifically binding to the target analyte. In this type of assay, thedetection conjugates will bind to the capture molecules initially. If asample containing a target analyte is mixed with these initialcomplexes, the unlabeled or free target analyte in the sample willcompete with the detection conjugates for binding to the capturemolecules. The change in optical signal in this type of assay willresult from the displacement of the metallic nanostructures in thedetection conjugates from the metallic nanolayer surface, which willproportionately reduce the wavelength shift in the peak absorptionwavelength

A test sample can be any type of liquid sample, including biologicalsamples or extracts prepared from environmental or food samples. In oneparticular embodiment, the test sample is a biological sample.Biological samples include, but are not limited to, whole blood, plasma,serum, saliva, urine, pleural effusion, sweat, bile, cerebrospinalfluid, fecal material, vaginal fluids, sperm, ocular lens fluid, mucous,synovial fluid, peritoneal fluid, amniotic fluid, biopsy tissues,saliva, and cellular lysates. The biological sample can be obtained froma human subject or animal subject suspected of having a diseasecondition, such as cancer, infectious diseases (e.g., viral-,bacterial-, parasitic- or fungal-infections), cardiovascular disease,metabolic disease, autoimmune disease etc. The biological sample canalso be obtained from a healthy subject (e.g. human or animal)undergoing a routine medical check-up.

In some embodiments of the methods, the test sample is mixed with theplurality of detection conjugates and the mixture is subsequentlybrought into contact with the metallic nanolayer surface containing theimmobilized capture molecules. In other embodiments, the test sample iscontacted with the metallic nanolayer surface containing the immobilizedcapture molecules and the plurality of detection conjugates issubsequently added. In certain embodiments, the sample, the plurality ofdetection conjugates, and the metallic nanolayer surface containing theimmobilized capture molecules are brought into contact at the same time.For instance, contact of the sample with both reagents simultaneouslymay occur in the rotor-format detection devices described above.

Any of the analyte detection devices described above can be used in thedetection methods of the present invention. Accordingly, the variousmetallic nanolayer surfaces, capture molecules, and detection conjugatesdescribed herein are suitable for use in the detection methods. Forinstance, in some embodiments of the methods, the surface containing ametallic nanolayer is a chip, a well, a cuvette, or a bead. In certainembodiments of the methods, the surface containing a metallic nanolayeris the walls and bottom of a cuvette incorporated into or adapted foruse with a centrifugal rotor. In these and other embodiments, themetallic nanolayer on the surface is a metallic film, such as a goldfilm. In other embodiments of the methods, the metallic nanolayer on thesurface comprises a plurality of metallic nanostructures immobilized onthe surface, such as gold nanostructures.

In certain embodiments of the detection methods, the detectionconjugates comprise composite metallic nanostructures coupled to bindingpartners or target analytes. As described herein, composite metallicnanostructures comprise at least two noble metals or transition metals.In some embodiments of the methods, the composite metallicnanostructures comprise at least two metals selected from gold, silver,copper, platinum, palladium, ruthenium, rhodium, osmium, iridium,titanium, chromium, cadmium, zinc, iron, cobalt, and nickel. In otherembodiments of the methods, the composite metallic nanostructurescomprise at least two metals selected from gold, silver, copper,platinum, palladium, cadmium, iron, nickel, and zinc. In one particularembodiment, the composite metallic nanostructures comprise gold andsilver. In another embodiment, the composite metallic nanostructurescomprise gold and copper. In yet another embodiment, the compositemetallic nanostructures comprise silver and copper. The compositemetallic nanostructures used in the methods of the invention can includea number of different geometries, such as spherical nanoparticles,pyramidal nanoparticles, hexagonal nanoparticles, nanotubes, nanoshells,nanorods, nanodots, nanoislands, nanowires, nanodisks, nanocubes, orcombinations thereof.

In certain embodiments, the composite metallic nanostructures used inthe methods of the invention are alloys of a first metal and a secondmetal. In some embodiments, the composite metallic nanostructures usedin the methods of the invention comprise a core of a first metal and acoating of a second metal. In particular embodiments, the compositemetallic nanostructures comprise a silver core and a gold coating. Inother embodiments, the composite metallic nanostructures comprise acopper core and a gold coating. In another embodiment, the core issilver and the coating is copper. In some embodiments, each of thecomposite metallic nanostructures comprises a dielectric core (e.g.silicon dioxide, gold sulfide, titanium dioxide, silica, andpolystyrene), a first coating of a first metal, and a second coating ofa second metal. In one particular embodiment of the detection methods,the core is silica, the first coating (i.e. inner coating) is a silvercoating, and the second coating is a gold coating (i.e. outer coating).In another embodiment, the core is silica, the first coating (i.e. innercoating) is a copper coating, and the second coating is a gold coating(i.e. outer coating).

The detection methods of the invention may be used to determinequalitative or quantitative amounts of a target analyte. Such methodsare particularly useful for determining the approximate amount of atarget analyte in a sample, which can be used inter alia to diagnosecertain medical conditions or evaluate the efficacy of a drug therapy.In one embodiment, the quantity of a target analyte can be determined byestablishing a standard curve for the particular analyte by measuringchanges in optical signals from the metallic nanolayer surface asdescribed herein for samples with a known quantity of target analyte;determining the optical signal change for a test sample; and comparingthe optical signal change for the test sample to the values obtained forthe standard curve. In some embodiments, determining the quantity of acomplex between a first reagent and a second reagent comprises comparingthe absorbance ratio and/or reaction rate from a test sample to theabsorbance ratio and/or reaction rate from one sample with a knownquantity of complex, thereby determining the quantity of the complex inthe test sample. The quantitative values obtained from test samples maybe compared to pre-determined threshold values, wherein saidpre-determined threshold values are indicative of either an abnormal ornormal level of the target analyte.

The detection methods of the present invention provide a highlysensitive technique for detecting minute quantities of a target analytein a sample. As demonstrated by the working examples, amplification ofplasmon resonance-based signals from gold nanolayer surfaces can beachieved with gold nanostructure conjugates such that nanogramquantities of target analyte can be detected in a sample. Thus, in oneembodiment of the methods, the presence of nanogram quantities of atarget analyte is detected. The inventors have surprisingly found thatsignificantly greater amplification of plasmon resonance-based signalsfrom gold nanolayer surfaces can be achieved with composite metallicnanostructure conjugates. Use of gold-coated silver nanostructuresconjugated to an analyte-specific antibody enabled the detection ofpictogram quantities of the target analyte, which is a 1000-foldincrease in sensitivity as compared to that obtained with goldnanostructure conjugates. See Example 3. Accordingly, in someembodiments of the methods, the presence of picogram quantities of thetarget analyte is detected. In other embodiments of the methods, thepresence of femtogram quantities of the target analyte is detected.Greater sensitivities may be obtained by altering the composition and/orshape of the composite metallic nanostructures and/or metallic nanolayersurface.

When incident light is applied to metallic nanostructures, conductionband electrons in the metal oscillate collectively at the same frequencyof the incident electromagnetic wave. As a result of these resonanceoscillations, the nanostructures strongly absorb and scatter light at aspecific wavelength range. For metallic nanostructures comprising nobleor transition metals, this wavelength range is in theultraviolet-visible-infrared spectrum depending on the particularcomposition of the nanostructures. Thus, light sources for applyingelectromagnetic energy suitable for use in the methods of the inventioncan include any source that may apply a wavelength range within theultraviolet-visible spectrum or ultraviolet-visible-infrared spectrum,including arc lamps and lasers. In some embodiments, the light sourcemay be equipped with a monochromator so that specific wavelengths oflight may be applied to the metallic nanolayer surface.

The optical properties of the metallic nanolayers and nanostructuresdepend on their size, shape, and composition. For instance, solid goldnanoparticles have an absorption peak wavelength (λ_(max)) from about515 nm to about 560 nm depending on particle size. Gold sphericalnanoparticles having a 30 nm diameter maximally absorb at about 520 nmwith λ_(max) shifting to longer wavelengths as particle diameterincreases. Silver and copper particles have a λ_(max) in theultra-violet/blue or red region (e.g., from about 350 nm to about 500nm) with increasing particle diameter causing a shift in λ_(max) tolonger wavelengths. Metallic nanorods have a transverse λ_(max1) and alongitudinal λ_(max2). Alloys of different metals typically exhibitabsorption peaks in an intermediate range between the absorption peaksof the comprising metals. For example, nanostructures comprising a 50/50alloy of gold and silver exhibit a λ_(max) of about 470 nm withincreasing amounts of gold causing a shift in the absorption peak tolonger wavelengths. The sensitivity of the LSPR signals to changes inthe local medium refractive index can be modified by changing the shapeor geometry of the nanostructures. For instance, nonspherical particles(e.g. nanoprisms, nanorods, nanoshells, etc.) have increased LSPRsensitivities as compared to spheres. In some embodiments, the opticalproperties (e.g. absorption/scattering at particular wavelengths) aretailored to a particular application by varying the size, shape, orcomposition of the metallic nanolayer deposited on the surface or themetallic nanostructures employed in the detection conjugates.

The interaction between the incident light and the metallic nanolayersurface can be monitored as reflected light or transmitted light. Theamount of the incident light that is absorbed or scattered can bemeasured as an absorption spectrum in a reflection mode or theabsorption spectrum in a transmission mode. In some embodiments, theoptical signal measured from the metallic nanolayer can be an opticalreflection, an absorbance spectrum, a scattering spectrum, and/or anemission spectrum.

The plasmon coupling between the metallic nanolayer and the metallicnanostructures in the detection conjugates resulting from complexformation between the binding partners, target analyte, and capturemolecules produces a change in the localized surface plasmon resonancespectrum of the metallic nanolayer. For instance, such changes caninclude an increased optical extinction, an increased opticalreflection, and/or increased scattering and/or emission signal. In someembodiments, the change in optical signal indicative of the presence ofthe target analyte in the sample includes a shift, increase or decreasein optical scattering or a combination of these features. In certainembodiments, the change in optical signal indicative of the presence ofthe target analyte in the sample is a spectral peak wavelength shift. Inone embodiment, the wavelength shift in the optical spectral peak may bea red shift (e.g., a shift to a longer wavelength) within a 200 nm to1200 nm spectral window. In another embodiment, the wavelength shift inthe optical spectral peak may be a blue shift (e.g., a shift to ashorter wavelength) within a 200 nm to 1200 nm spectral window. Thechanges in optical signals can be measured at a particular time pointfollowing a set reaction period. Additionally or alternatively, changesin the optical signal over the reaction period (e.g. ratedeterminations) may be measured. Both types of measurements can be usedfor either qualitative or quantitative analysis of a target analyte.

Various means for measuring optical signals at different wavelengths andacquiring extinction, scattering, or emission spectra are known in theart. Any spectrophotometric or photometric instruments are suitable foruse in the disclosed methods. Some non-limiting examples include platereaders, Cobas Fara analyzers, and Piccolo Xpress® and Vetscan analyzers(Abaxis, Inc., Union City, Calif.), optic fiber readers (e.g.,LightPath™ S4 (LamdaGen, Menlo Park, Calif.)), SPR instruments (e.g.,Biacore instruments available from GE Healthcare), centrifugal analyzersfrom Olympus, Hitachi etc.

The present invention also includes an assay complex comprising (i) adetection conjugate that comprises a composite metallic nanostructurecoupled to a binding partner, (ii) a target analyte, and (iii) ametallic nanolayer-coated bead on which a capture molecule isimmobilized, wherein the binding partner in the detection conjugate isbound to a first epitope on the target analyte and the capture moleculeis bound to a second epitope on the target analyte, thereby forming acomplex comprising the detection conjugate, target analyte, and thecapture molecule. In some embodiments, the assay complex is containedwithin a cuvette adapted for use with a centrifugal rotor. In otherembodiments, the assay complex is contained within a reaction chamber ina centrifugal rotor or disc.

The binding partner and capture molecule in the assay complex can be anytype of molecule described above, including haptens and other smallmolecules, drugs, hormones, biological macromolecules such as antibodiesor fragments thereof (e.g., Fv, Fab, (Fab)₂, single chain, CDR etc.),antigens, receptors, ligands, polynucleotides, aptamers, polypeptides,polysaccharides, lipopolysaccharides, glycopeptides, lipoproteins, ornucleoproteins. In one embodiment, the binding partner is an antibodyand the capture molecule is a different antibody.

The metallic nanolayer and composite metallic nanostructures aredescribed in detail above. In one embodiment, the metallic nanolayercoating the bead (e.g. plastic or glass bead) is a gold nanolayer. Inanother embodiment, the metallic nanolayer coating the bead is a silvernanolayer. The bead is preferably less than 0.5 cm, but greater than 0.1mm. In certain embodiments, the composite metallic nanostructures aregold-coated silver nanostructures. In other embodiments, the compositemetallic nanostructures are gold-coated copper nanostructures. In stillother embodiments, the metallic nanostructures comprise gold doped withsilver, copper ions or both of these ions.

Any type of target analyte can be detected using the methods, devices,and assay complexes of the present invention, particularly those thatare significant in the diagnoses of diseases. A target analyte caninclude, but is not limited to, a protein, enzyme, antigen, antibody,peptide, nucleic acid (RNA, DNA, mRNA, miRNA), hormone, glycoprotein,polysaccharide, toxin, virus, virus particle, drug molecule, hapten, orchemical. In some embodiments, the target analyte is a marker or antigenassociated with an infectious disease in humans and/or animals. In otherembodiments, the target analyte is a marker or antigen associated with aparticular physiological state or pathological condition.

In certain embodiments, the target analyte is a pathogenic antigen orantibody to a pathogenic antigen. For instance, the pathogenic antigencan be a viral antigen (e.g., feline leukemia virus, canine parvovirus,foot and mouth virus, influenza virus, hepatitis a, b, c virus, HIVvirus, human papilloma virus, Epstein Barr virus, rabies virus, etc.), abacterial antigen (e.g., Ehrlichia, Borrelia, Anaplasma, Anthrax,Salmonella, Bacillus, etc.), a fungal antigen, or parasitic antigen(e.g., canine heartworm, Giardia lamblia, plasmodium falciparum, Africantrypanosomiasis, Trypanosoma brucei, etc.). In other embodiments, thetarget analyte is a disease-related antigen or antibody to adisease-related antigen. Disease-related antigens include, but are notlimited to, cancer-related antigens or markers (e.g., PSA, AFP, CA125,CA15-3, CA19-9, CEA, NY-ESO-1, MUC1, GM3, GD2, ERBB2, etc.),cardiovascular disease-related antigens or markers (e.g., troponin,C-reactive protein, brain natriuretic peptide, CKMB, fatty acid bindingprotein, etc.,), metabolic-related antigens or markers (e.g., thyroidstimulating hormone, thyroxine, leptin, insulin), or autoimmunedisease-related antigens or markers (e.g., auto-antibodies). In certainembodiments, the target analyte is an inflammatory antigen or marker(e.g., C-reactive protein, MRP14, MRP8, 25F9, etc.). In otherembodiments, the target analyte is a pregnancy-related antigen or marker(e.g., a fetal antigen, human chorionic gonadotropin).

The present invention also provides a method for preparing compositemetallic nanostructures. In one embodiment, the method comprisespreparing a first solution comprising a mixture of a polymer andchloroauric acid, preparing a second solution comprising silver orcopper nanostructures, and incubating the first solution with the secondsolution for a period of time, wherein the resulting mixture comprisesgold-coated silver nanostructures or gold-coated copper nanostructures.The resulting mixture preferably has a peak absorbance of about 515 nmto about 670 nm, or about 520 nm to about 560 nm. In one embodiment, theresulting mixture has a peak absorbance of about 530 nm.

The polymer used in the preparation of the first solution can be any oneof polyvinylpyrrolidone, polyvinyl alcohol, polyacrylate, polyethyleneglycol, polyethyleneimine, polyaspartic acid, polyglutamic acid, variousgums, gelatin or mixed polymers comprising any of the foregoing. In oneparticular embodiment, the polymer is polyvinylpyrrolidone. Differenttypes of coated nanostructures can be obtained by varying the molecularweight of the polymer. Suitable molecular weight ranges of the polymerinclude from about 5,000 Daltons to about 150,000 Daltons, about 10,000Daltons to about 100,000 Daltons, from about 20,000 Daltons to about80,000 Daltons. In some embodiments, the polymer has a molecular weightless than 50,000 Daltons. In other embodiments, the polymer has amolecular weight less than 20,000 Daltons. In certain embodiments, thepolymer has a molecular weight of about 10,000 Daltons.

The characteristics of the gold coating can be controlled by adjustingthe concentration ratio of polymer to chloroauric acid. For instance,the concentration ratio of polymer to chloroauric acid is from about100:1 to about 1:100, from about 2:1 to about 5:1, or from about 1.5:1to about 8:1. In some embodiments, the concentration ratio of polymer tochloroauric acid is 1:1. Suitable concentrations of polymer include, butare not limited to, about 0.1% to about 20% wt/wet in water or ethanol.Suitable concentrations of chloroauric acid include, but are not limitedto, about 0.001 M to about 1.0 M, about 0.010 M to about 0.500 M, andabout 0.050 M to about 0.100 M.

The coating efficiency and thickness can also be affected by the pH andhalide content of the coating solution (i.e. first solution). In certainembodiments, the pH of the solution is kept in a range from about 3 toabout 14. The halide content of the solution is, in some embodiments,less than 150 mM. In other embodiments, the halide content of thesolution is in the range of about 0 to about 50 mM.

Methods of preparing solutions of silver and copper nanostructures areknown to those of skill in the art. For instance, the second solutioncomprising silver or copper nanostructures can be prepared by any of themethods described in U.S. Patent Publication No. 2012/0101007, U.S.Patent Publication No. 2014/0105982, or U.S. Patent Publication No.2013/0230717, each of which is hereby incorporated by reference in itsentirety. In one embodiment, the second solution comprising silver orcopper nanostructures is prepared by mixing a silver or copper sourcewith a reducing agent. A suitable silver source includes a silver salt,such as silver nitrate. Suitable copper sources include copper (II)sulfate, copper (II) chloride, copper (II) hydroxide and copper (II)nitrate, copper (II) acetate and copper (II) trifluoroacetate. Reducingagents that can be reacted with the silver or copper sources to form thenanostructures can include glucose, ascorbic acid, sodium borohydride,and alkaline solutions (e.g. pH greater than 7.5) of polymers such asPVP. In certain embodiments, the reducing agent is ascorbic acid. Thedesired shape and optical spectral peak of the silver nanostructures orcopper nanostructures can be attained by adjusting the ratios orconcentrations of reactants as known to those of ordinary skill in theart. By way of example only, high concentrations of the reducing agentcan result in pentagonal- and bipyramidal-shaped nanostructures, whereaslow concentrations of the reducing agent can result in elongatednanowires or tubes. Depending on the particular shapes of thenanostructures, the second solution comprising silver or coppernanostructures may have a peak absorbance from about 550 nm to about1000 nm, from about 600 nm to about 700 nm, from about 630 nm to about680 nm, from about 750 nm to about 850 nm, from about 900 nm to about940 nm, from about 580 nm to about 620 nm, or from about 550 nm to about750 nm. In certain embodiments, the second solution comprising silvernanostructures has a peak absorbance of about 600 nm (i.e. 595 nm to 605nm, inclusive). In some embodiments, the second solution comprisingcopper nanostructures has a peak absorbance of about 585 nm (i.e. 580 nmto 590 nm, inclusive). In some embodiments the peak absorbance of asolution comprising copper nanostructures is greater (i.e. red-shifted)than the peak absorbance of a solution comprising silver nanostructuresof a similar size and shape.

In some embodiments, the incubation period of the first solution withsecond solution is at least 12 hours. In other embodiments, theincubation period of the first solution with second solution is greaterthan 24 hours, preferably greater than 48 hours, more preferably atleast 72 hours. Changes in the peak absorbance of the reaction mixturecan be monitored during the incubation period to adjust the incubationtime accordingly. For example, shifts of the peak absorbance to shorterwavelengths, for instance in the 520 nm to 550 nm region, can indicatethat the gold-coated nanostructures have stabilized. In certainembodiments, stability of the resulting nanostructures to sodiumchloride (e.g., 0.25-1M) is used to indicate a proper coating of thenano structures.

In certain embodiments, the present invention provides methods ofsynthesizing nanostructures having optical densities greater than about50/mL. In one embodiment, the methods comprise mixing a polymer asdescribed herein with chloroauric acid, stirring the mixture at a settemperature for a first period of time, adding ascorbic acid to themixture, and incubating the mixture for a second period of time. Thesize and shape of the nanostructures is dictated by the concentrationratio of polymer to chloroauric acid and the temperature and time ofincubation. The concentrations of polymer and chloroauric acid can be inthe ranges described above. The temperature can be adjusted based on thesize and shape of the nanostructures desired, but may be in the range ofabout 4° C. to about 100° C. Similarly, the incubation period (i.e.first period of time) can be adjusted based on the desired properties ofthe nanostructures, but may range from about 15 minutes to one day.

In some embodiments, about 0.1 to 1 part of ascorbic acid (e.g. about 1to 5 M) is added to the mixture following the first incubation period.The second incubation period following addition of the ascorbic acid maybe from about 1 to about 24 hours. Without being bound by theory,addition of ascorbic acid provides a substantial increase in thequantity of nanostructures produced.

In certain embodiments, the methods further comprise adding or dopingthe mixture with about 1 to about 100 parts of gold chloride (e.g. about0.001 M to 1M) or silver nitrate (e.g. about 0.001 M to 1M) or othermetal (e.g. noble metal, transition metal, alkali metal, or lanthanide).This doping step can further increase the resonance intensity of theresulting nanostructures. In some embodiments, the gold chloride, silvernitrate, or other metal is added to the mixture before ascorbic acid isadded to the reaction. In other embodiments, the gold chloride, silvernitrate, or other metal is added to the mixture following the additionof ascorbic acid. The order of addition of the metal and ascorbic acidmay be adjusted to tailor the resulting nanostructures to a desiredshape and size.

This invention is further illustrated by the following additionalexamples that should not be construed as limiting. Those of skill in theart should, in light of the present disclosure, appreciate that manychanges can be made to the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

All patent and non-patent documents referenced throughout thisdisclosure are incorporated by reference herein in their entirety forall purposes.

Examples Example 1 Amplification of LSPR Signals with GoldNanoparticle-Conjugated Analyte

An analyte detection system was prepared by providing a plastic chip onwhich was deposited a gold nanolayer film. Human IgG proteins (100μg/ml) were immobilized to the gold nanolayer film to create the sensorsurface. A control sensor was constructed by immobilizing bovine serumalbumin to the gold nanolayer film. The two types of sensor surfaceswere positioned within an instrument equipped with light emitting andlight collecting fibers that shine light on the gold nanolayer surfaceand collect the light reflected back from the surface.

A sample containing free protein A (10 μg/ml) was contacted with the twotypes of sensor surfaces and the changes in the reflectance spectra weremeasured. As shown in FIG. 1, introduction of free protein A to thesensor containing immobilized human IgG does not produce a significantvisible change in the reflectance spectrum of the gold nanolayer film asmeasured by a shift in the peak wavelength.

The sensor surfaces were regenerated by treatment with 1 mM hydrochloricacid and sample containing protein A conjugated to colloidal goldnanoparticles (CGC) at two different concentrations (3.5 μg/ml and 0.175μg/ml) were contacted with the sensor surfaces. The change in thereflectance spectrum of the gold nanolayer surface was accentuated whenthe protein A (i.e. target analyte) was conjugated to colloidal goldnanoparticles. Specifically, 3.5 μg/ml of protein A-CGC produced agreater shift in peak wavelength than 10 μg/ml of unlabeled protein A.See FIG. 1, sensor 2. The amplification of the plasmon resonance signalwas sufficiently great to enable detection of nanogram concentrations ofprotein A-CGC. See FIG. 1, sensor 3. The changes in the reflectancespectrum of the BSA sensor represent non-specific binding of the proteinA molecules to the sensor surface and are significantly smaller than thechanges induced by specific binding of the protein A molecules to theimmobilized IgG molecules.

The results of this initial experiment show that considerableamplification of the changes in localized surface plasmon resonancesignals induced by binding events at a metallic nanolayer surface can beachieved by coupling the target analyte to colloidal gold nanoparticles.Nearly a 60-fold improvement in sensitivity is observed with nanogramquantities of analyte being detected.

Example 2 Amplification of LSPR Signals in a Sandwich Assay

This example describes a series of experiments designed to evaluatewhether amplification of localized surface plasmon resonance signalswith gold nanoparticle conjugates could also be achieved in a sandwichassay format in which the target analyte is not directly conjugated tothe gold nanoparticles. A gold nanolayer chip surface was prepared asdescribed in Example 1. The C7 antibody against C-reactive protein (CRP)(100 μg/ml) was immobilized to the gold nanolayer film deposited on thechip surface to create the anti-CRP sensor. The C6 antibody, whichrecognizes a distinct, non-overlapping epitope of CRP than the C7antibody, was conjugated to colloidal gold nanoparticles (C6-CGC) forsome experiments or used in an unlabeled form for other experiments.

In a first series of experiments, a sample containing one of threedifferent concentrations of CRP (1 ng/ml, 10 ng/ml, or 100 ng/ml) wasincubated with the anti-CRP sensor for 15 to 20 minutes and changes inthe reflectance spectrum of the gold nanolayer were monitored. As shownin FIG. 2, very minimum peak shift was observed upon binding of the CRPto the immobilized C7 anti-CRP antibody on the sensor surface.Subsequent exposure of the sensor surface to unlabeled C6 anti-CRPantibody (1 μg/ml) did not result in significant further peak shifts.See FIG. 2. Similarly, subsequent exposure of the sensor surface to 3μg/ml of C6-CGC did not produce any further changes in the reflectancespectrum, indicating that the bound CRP molecules were likely saturatedwith unlabeled C6 antibody. See FIG. 2.

In a second series of experiments, a sample containing one of threedifferent concentrations of CRP (1 ng/ml, 10 ng/ml, or 100 ng/ml) wasincubated with the anti-CRP sensor for 15 to 20 minutes. Two differentconcentrations of C6-CGC (1 μg/ml and 3 μg/ml) were subsequentlyintroduced and changes in the reflectance spectrum were measured (FIG. 3and panel A of FIG. 4). The results show that conjugation of the C6anti-CRP antibody to gold nanoparticles amplifies the peak wavelengthshift as compared to unlabeled C6 antibody. Increasing concentrations ofC6-CGC produce a dose-dependent shift in peak wavelength. However, thesignal difference between 1 ng/ml and 10 ng/ml was small (panel B ofFIG. 4).

In a third series of experiments, the effect of analyte incubation timeon signal development was evaluated. The anti-CRP sensor was contactedwith sample containing 0 ng/ml, 10 ng/ml, or 100 ng/ml CRP and 3 μg/mlC6-CGC was immediately introduced without any analyte incubation time ora wash. As shown in FIGS. 5 and 6, shorter analyte incubation timeresults in smaller peak wavelength shifts.

The results of these three sets of experiments show that amplificationof LSPR signals can be achieved with gold nanoparticle conjugates in asandwich assay format. An enhanced signal shift is observed when thedetector antibody is labeled with colloidal gold particles as comparedto unlabeled antibody, thereby allowing for detection of nanogramconcentrations of analyte.

Example 3 Enhanced Signal Amplification with Gold-Coated SilverNanostructures

To examine whether varying the type of metal used to label the bindingpartners affected the amplification of LSPR signals, composite metalnanostructures were prepared. Specifically, gold-coated silvernanostructures were prepared as follows. Silver nanostructures wereprepared by adding 50.0 mL of deionized H₂O, 500.0 μL Trisodium Citrate(75 mM), 200 μL AgNO₃ (200 mM), and 500.0 μL H₂O₂ (27%) while stirringvigorously at room temperature. A 500 μL aliquot of NaBH₄ (200 mM) wasthen rapidly injected into the aqueous solution causing a color changeto light yellow. Over a period of several minutes the color continued tochange from dark yellow to red to purple and finally stabilizing atblue. The UV/Vis spectra determined the peak absorbance of the solutionto be at 604.5 nm.

A gold coating was added to the silver nanostructures by adding 5.0 mLof the blue solution to a mixture of 50 μL of Polyvinylpyrrolidone (PVPMW≈10,000 20% in ethanol) and 50 μl, of HAuCl₄ (20 mM). After 72 hoursof incubation time the sample became a dark red color and had a peakabsorbance at 534.5 nm. The nanoparticles were washed twice bycentrifugation at 20,000 rpm for 20 minutes and resuspended in 2.0 mL ofdeionized H₂O. The solution had a deep red color, absorption peak at530.3 nm, and a total absorbance of 15.0 OD units.

Conjugation of gold-coated silver nanostructures (Au@AgNPs) to the C6anti-CRP antibody was performed by adding 600.0 μL of Au@AgNPs and 20.0μL C6 anti-CRP antibody (8.0 mg/mL) to 880.0 μL deionized H₂O bringingthe final antibody concentration to 17.8 μg/mL/OD. After a 2 hourincubation period at 4° C., the sample was centrifuged at 30,000 g for20 minutes and resuspended in 1.5 mL of a blocking solution containingBSA (10 mg/mL) in PBS. The Au@AgNPs conjugated to anti-CRP C6 antibodywere stored at 4° C. until further use.

The anti-CRP gold nanolayer sensor was prepared as described in Example2 and had peak absorption at 530 nm. A control sensor containing thegold nanolayer without any immobilized antibody was also prepared. Thesensors were equilibrated with 100 μL PBS.

100 μL C6 anti-CRP antibody conjugated to Au@AgNPs diluted to 1.5 OD inPBS was premixed for 1 minute with 1, 10, or 500 pg/mL CRP antigen. Themixture was then brought into contact with the anti-CRP or controlsensor surface and changes in the reflectance spectrum of the goldnanolayer surface were measured. The results show that the gold-coatedsilver nanostructures enhanced the peak wavelength shift induced bybinding of the CRP-antibody complex to the sensor surface (FIG. 7).Detection of 1 pg/mL of CRP antigen was possible with the gold-coatedsilver nanostructures, which is a 1000-fold improvement in sensitivityas compared to that obtained with gold nanoparticles. At higherconcentrations of antigen the binding sites are saturated and no furthershifts occur.

The results of this experiment demonstrate the significantly enhancedamplification of LSPR signals from a metallic nanolayer surface achievedwhen composite nanostructures, such as gold-coated silvernanostructures, are used to label analyte binding partners.

Example 4 Synthesis of High Optical Density Nanostructures

Gold nanoparticles were prepared by mixing the following reagents in afinal volume of 1 ml in the indicated order: 0.1 ml of 1% PVP-10 (1%wt/wt), 0.2 ml of 0.1M gold chloride, 0.1 ml of 5N NaOH, 0.4 ml of waterand 0.2 ml of 1M ascorbic acid. The reaction mixture was mixed aftereach addition. The spectroscopic measurements indicated that thereaction was mostly complete after 24 hours at room temperature. Thisprotocol yielded spherical gold nanoparticles exhibiting the LSPR peakaround 535 nm and the corresponding optical density of about 80 per ml.Layering with additional gold or silver was done by adding silvernitrate or gold chloride to the preformed gold nanoparticles. Excessreagents were removed by centrifugation at 30,000 g for 1-2 hours.

In a separate reaction, 0.05 ml of 20% PVP (wt/wt) was mixed with 0.25ml water, 0.1 ml of 5N NaOH, 0.1 ml of 1 M sodium citrate, 0.5 ml of0.1M gold chloride and 1 ml of 1M ascorbic acid. This protocol resultedin immediate formation of colloidal gold particles at an OD of about90/ml with LSPR peak at ˜525 nm. A linear correspondence was observedbetween final OD and the concentration of gold between 2.5 mM gold and25 mM gold in the final reaction mixture.

It is understood that the disclosed invention is not limited to theparticular methodology, protocols and materials described as these canvary. It is also understood that the terminology used herein is for thepurposes of describing particular embodiments only and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An analyte detection device comprising: a plurality of detectionconjugates, wherein the conjugates comprise composite metallicnanostructures coupled to binding partners that are capable ofspecifically binding to a target analyte; a surface containing ametallic nanolayer; and a plurality of capture molecules, wherein thecapture molecules are immobilized on the metallic nanolayer and arecapable of specifically binding to the target analyte.
 2. An analytedetection device comprising: a plurality of detection conjugates,wherein the conjugates comprise composite metallic nanostructurescoupled to target analytes; a surface containing a metallic nanolayer;and a plurality of capture molecules, wherein the capture molecules areimmobilized on the metallic nanolayer and are capable of specificallybinding to the target analytes.
 3. The analyte detection device of claim1, wherein the composite metallic nanostructures comprise at least twometals selected from gold, silver, copper, platinum, palladium, cadmium,iron, nickel, and zinc.
 4. The analyte detection device of claim 1,wherein each of the composite metallic nanostructures comprises a coreof a first metal and a coating of a second metal.
 5. The analytedetection device of claim 4, wherein each of the composite metallicnanostructures comprises a gold coating and a sliver core.
 6. Theanalyte detection device of claim 1, wherein each of the compositemetallic nanostructures is an alloy of a first metal and a second metal.7. The analyte detection device of claim 1, wherein the compositemetallic nanostructures are spherical nanoparticles and have a diameterof about 5 nm to about 200 nm.
 8. The analyte detection device of claim1, wherein the composite metallic nanostructures are sphericalnanoparticles and have a diameter of about 10 nm to about 100 nm.
 9. Theanalyte detection device of claim 1, wherein the composite metallicnanostructures are nanoplates with an edge length of about 10 nm toabout 800 nm and a thickness of about 1 nm to about 100 nm.
 10. Theanalyte detection device of claim 1, wherein the plurality of detectionconjugates is in the form of a lyophilized pellet or bead.
 11. Theanalyte detection device of claim 1, wherein the surface is a chip, awell, a bead, or a wall, cover, and/or bottom of a cuvette.
 12. Theanalyte detection device of claim 1, wherein the metallic nanolayer is ametallic film.
 13. The analyte detection device of claim 12, wherein themetallic film comprises gold, silver, copper, platinum, palladium,cadmium, zinc or a composite thereof.
 14. The analyte detection deviceof claim 12, wherein the metallic film comprises gold.
 15. The analytedetection device of claim 1, wherein the metallic nanolayer comprises aplurality of metallic nanostructures immobilized on the surface.
 16. Theanalyte detection device of claim 15, wherein the plurality of metallicnanostructures comprise gold, silver, copper, platinum, palladium,cadmium, zinc or a composite thereof.
 17. The analyte detection deviceof claim 15, wherein the plurality of metallic nanostructures are goldnanostructures.
 18. The analyte detection device of claim 1, wherein thecomposite nanostructures have a geometry selected from sphericalnanoparticles, pyramidal nanoparticles, hexagonal nanoparticles,nanoshells, nanotubes, nanorods, nanodots, nanoislands, nanowires, orcombinations thereof.
 19. The analyte detection device of claim 1,wherein the binding partners and/or capture molecules are antibodies,antigens, polypeptides, polynucleotides, nucleoproteins, aptamers,ligands, receptors, or haptens.
 20. The analyte detection device ofclaim 1, wherein the binding partners are antibodies that recognize afirst epitope of a target analyte and the capture molecules aredifferent antibodies that recognize a second epitope of a targetanalyte.
 21. The analyte detection device of claim 2, wherein thecapture molecules are antibodies, antigens, polypeptides,polynucleotides, nucleoproteins, aptamers, ligands, receptors, orhaptens.
 22. The analyte detection device of claim 1, wherein the targetanalyte is a marker or antigen associated with an infectious disease,physiological state, or pathological condition.
 23. The analytedetection device of claim 1, wherein the target analyte is canineheartworm, feline leukemia virus, canine parvovirus, C-reactive protein,Giardia lamblia, Ehrlichia antigen or antibody, Borrelia antigen orantibody, Anaplasma antigen or antibody, a cancer antigen, a cardiacmarker antigen, thyroid stimulating hormone, thyroxine, troponin, orbrain natriuretic peptide.
 24. A method of detecting a target analyte ina sample comprising: mixing the sample with a plurality of detectionconjugates, wherein the conjugates comprise composite metallicnanostructures coupled to binding partners that are capable ofspecifically binding to the target analyte if present in the sample toform analyte-detection conjugate complexes; contacting the mixture witha surface containing a metallic nanolayer, wherein a plurality ofcapture molecules are immobilized on the metallic nanolayer and arecapable of specifically binding to the target analyte if present in thesample; exposing the surface to a light source at a wavelength rangewithin the ultraviolet-visible-infrared spectrum; and measuring anoptical signal from the surface, wherein a change in the optical signalindicates the presence of the target analyte in the sample.
 25. Themethod of claim 24, wherein the optical signal is reflectance, anabsorbance spectrum, scattering spectrum, or an emission spectrum. 26.The method of claim 24, wherein the change in the optical signalcomprises a spectral peak wavelength shift.
 27. The method of claim 24,wherein the presence of nanogram quantities of the target analyte isdetected.
 28. The method of claim 24, wherein the presence of picogramquantities of the target analyte is detected.
 29. The method of claim24, wherein the presence of femtogram quantities of the target analyteis detected.
 30. The method of claim 24, wherein the surface is thewalls and bottom of a cuvette incorporated into a centrifugal rotor. 31.The method of claim 24, wherein the composite metallic nanostructurescomprise at least two metals selected from gold, silver, copper,platinum, palladium, cadmium, iron, nickel, and zinc.
 32. The method ofclaim 24, wherein each of the composite metallic nanostructurescomprises a core of a first metal and a coating of a second metal. 33.The method of claim 32, wherein each of the composite metallicnanostructures comprises a gold coating and a sliver core.
 34. Themethod of claim 24, wherein each of the composite metallicnanostructures is an alloy of a first metal and a second metal.
 35. Themethod of claim 24, wherein the metallic nanolayer is a metallic film.36. The method of claim 35, wherein the metallic film comprises gold.37. The method of claim 24, wherein the metallic nanolayer comprises aplurality of metallic nanostructures immobilized on the surface.
 38. Themethod of claim 37, wherein the plurality of metallic nanostructures aregold nanostructures.
 39. The method of claim 24, wherein the compositenanostructures have a geometry selected from spherical nanoparticles,pyramidal nanoparticles, hexagonal nanoparticles, nanotubes, nanoshells,nanorods, nanoislands, nanodots, nanowires, or combinations thereof. 40.A method for preparing composite metallic nanostructures comprising:preparing a first solution comprising a mixture of a polymer andchloroauric acid; preparing a second solution comprising silver orcopper nanostructures; and incubating the first solution with the secondsolution for a period of time, wherein the resulting mixture comprisesgold-coated silver nanostructures or gold-coated copper nanostructures.41. The method of claim 40, wherein the second solution comprises silvernanostructures and has a peak absorbance of about 550 to 750 nm.
 42. Themethod of claim 40, wherein the polymer is polyvinylpyrrolidone,polyvinyl alcohol, polyacrylate, polyethylene glycol orpolyethyleneimine.
 43. An assay complex comprising: a detectionconjugate comprising a composite metallic nanostructure coupled to abinding partner; a target analyte; and a metallic nanolayer-coated beadon which a capture molecule is immobilized, wherein the binding partnerin the detection conjugate is bound to a first epitope on the targetanalyte and the capture molecule is bound to a second epitope on thetarget analyte, thereby forming a complex comprising the detectionconjugate, target analyte, and the capture molecule.
 44. The assaycomplex of claim 43, wherein the binding partner is an antibody and thecapture molecule is a different antibody.
 45. The assay complex of claim43, wherein the metallic nanolayer is a metallic film.
 46. The assaycomplex of claim 45, wherein the metallic film comprises gold, silver,copper, platinum, palladium, cadmium, zinc or a composite thereof. 47.The assay complex of claim 46, wherein the metallic film comprises gold.48. The assay complex of claim 43, wherein the metallic nanolayercomprises a plurality of metallic nanostructures immobilized on thebead.
 49. The assay complex of claim 48, wherein the plurality ofmetallic nanostructures comprise gold, silver, copper, platinum,palladium, cadmium, zinc or a composite thereof.
 50. The assay complexof claim 49, wherein the plurality of metallic nanostructures are goldnanostructures.
 51. The assay complex of claim 43, wherein the compositemetallic nanostructure comprises at least two metals selected from gold,silver, copper, platinum, palladium, cadmium, iron, nickel, and zinc.52. The assay complex of claim 43, wherein the composite metallicnanostructure comprises a core of a first metal and a coating of asecond metal.
 53. The assay complex of claim 43, wherein the compositemetallic nanostructure comprises a gold coating and a sliver core. 54.The assay complex of claim 43, wherein the composite metallicnanostructure is an alloy of a first metal and a second metal.